Droplet-based microfluidics represents an alternative to using the conventional culturing of cells or cell-free systems in microtiter plates. Droplet-based microfluidic systems are characterized by low material consumption, a high throughput and a high sensitivity of the biological, medical or chemical analyses to be conducted. The microfluidic droplets are used as micro-reactors in a femtoliter to nanoliter volume range in numerous different applications.
EP 1 425 384 B1 describes a method for parallel culturing, culturing and analyzing single-cell microbial cultures, wherein nutrient substances, effectors and microbial metabolites are added to a microorganism population having a homogeneous or heterogeneous composition, and then 104 to 108 partial volumes from 0.1 nL to 1 μL are generated from the suspension and the resulting microcultures are incubated. The growth and the metabolic activities of the microcultures are determined using appropriate measurement methods.
WO 2009/048673 A2 relates to a method for culturing bacteria in a microfluidic system inside droplets that allows for detection of substances produced by said bacteria.
In WO 2012/103516 A1 a method is disclosed that allows for the detection of recombinantly produced compounds of cells, which are encapsulated by a hydrogel particle.
S. Köster et al. Lab Chip, 2008, 8, pp. 1110-1115 describes methods and devices for the encapsulation, incubation and manipulation of individual cells in aqueous droplets in a carrier fluid.
WO 2005/097969 A1 describes a microscale bioreactor for the cultivation of cells, which allows for a provision of oxygen to improve the culturing conditions. The system is not suited for the incubation of microfluidic droplets.
US 2008/0009027 A1 discloses an apparatus for the cultivation of cells in which oxygen may be enhanced by means of a gas-permeable membrane. The system is not suited for the incubation of microfluidic droplets.
In WO 2013/021035 A1, a microfluidic device and a method for cell-based assays are described, as is a method for using the microfluidic device for culturing cells. In particular, a “microfluidic apparatus” is claimed which has an area for culturing biological cells as well as a microfluidic channel for transporting a substance, wherein said culturing area and the channel are separated from one another by a wall that is permeable to the substance.
In US 2010/0124759 A1, the use of droplets for culturing and/or examining cells or other species is disclosed, wherein, for example, an examination is conducted to determine how different cell types react in a “microfluidic device” with different active substances, for example, an indicator or carbohydrate components such as sugar structures.
A disadvantage of the prior art is that so far only droplet incubations with greatly limited oxygen supply could be implemented. In addition, it has been found to be challenging to achieve a homogeneous and reproducible cell growth in droplets and to maintain it over long incubation times. An essential reason for this is the absence of homogeneous surrounding conditions with constant availability of the gases or gas mixtures used to a large number of droplets. In particular, concentration differences between the individual droplets with regard to the gases or gas mixtures dissolved in them, particularly oxygen, lead, for example, to inhomogeneities in the growth of cells or the production rates of metabolites. Differences in the surrounding can lead to the undesired formation of different phenotypes within the cells to be examined, whereby the results of a screening carried out based on these cells can become distorted or unusable.
Using the devices and methods available in the prior art, it is not possible to implement applications that require cell multiplication or the survival of cells over a prolonged time period, with reproducible results. Methods have indeed been described that are suitable for providing oxygen within the microfluidic droplets for short time periods. In the process, the high affinity of a carrier fluid for the gaseous oxygen is exploited. However, the disadvantage of this method is that the carrier fluid is not led cyclically through an incubator, and the number of the incubated droplets is seriously limited. As a result, only short incubation times are reached.
An essential technical challenge of droplet-based microfluidics consists in providing defined and reproducible conditions for culturing cells or cell-free systems that comprise isolated cell components or chemical components, for example, instead of viable cells. The production of these defined and reproducible conditions relates particularly to providing a homogeneous useful gas distribution in the microfluidic droplets. The term “useful gas” denotes, for the conceivable, primarily biological, applications of certain embodiments of the invention, the gas that is metabolized, for example, by the cell cultures inside the microfluidic droplets and converted into a waste gas in the process. For most cell cultures, the useful gas is oxygen or an air mixture which contains oxygen. However, such cell cultures, microorganisms or cell-free systems also exist that are preferably supplied with a useful gas that is different from oxygen or an air mixture.